Within this application several publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification, immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The medicinal leech Hirudo medicinalis has been used for centuries in the treatment of a variety diseases. The use of leeches for bloodletting reached its height in the eighteenth and early nineteenth centuries, and then began to give way to modern medicine. However, just when the leech was about to disappear from medical practice, new therapies using leeches emerged. Plastic surgeons have been using medicinal leeches to remove blood from post-operative occlusions, a procedure that increases the success of tissue transplants, reduction mammoplasty and the surgical re-attachment of amputated extremeties and digits by reducing the frequency of necrosis (1,2).
These new therapies created a major interest in medicinal leeches and efforts are directed at the isolation, characterization and synthesis of biologically active materials of leech saliva. The chemicals secreted in leech saliva, which may responsible for the success of the said operative procedures, are also being explored as possible therapeutic agents against several diseases, including thrombosis and atherosclerosis (3).
It has long been common knowledge that the host's blood continues to flow from the wound for a long time after the leech has ceased to feed. Indeed, it was shown a century ago (4) that extracts of the medicinal leech contain a substance, hirudin, which prevents blood clotting. Hirudin was isolated and characterized as a protein (5). The pharmacological effects of this potent anticoagulant in animals and in man (6, 7, 9) have also been extensively investigated. The advent of recombinant DNA technology opened the way to, and revived interest in the commercial production of hirudin.
Recombinant hirudin has been produced by major pharmaceutical companies and recent studies have shown that it effectively prevents thrombosis in several animal species and in man (9,10). The anticoagulant properties of hirudin are generally attributed to the inhibition of thrombin and consequently the blocking of fibrin formation. The beneficial effect of the medicinal leech of microsurgery, described above, could not, therefore, be due to hirudin, since thrombi in the microcirculation are usually due to platelet aggregation which is not inhibited by hirudin.
Apart from hirudin, saliva of the medicinal leech has been found to contain additional proteins, eglin, hyaluronidase, collagenase and apyrase (11, 12) and, in general, said publications (11, 12) disclose that leech saliva has platelet-aggregation inhibitory activity. Inhibition of platelet aggregation by leech saliva was also described in references (13) and (14). Platelet aggregation inhibitory activity has been found also in the saliva of a number of blood-sucking anthropods, such as the bug Rhodnius prolixus (15) and the tick Ixodes dammini (16).
Platelets have an important role in normal hemostasis amd in clinical disorders associated with thrombosis and atherosclerosis. Although sticky, they do not adhere to the normal intact endothelial surface of the vessel wall. Injury of the vascular endothelium causes rapid adhesion of circulating platelets on subendothelial structures, and soon other platelets from the circulation are activated by platelet-released products, ADP and thromboxane A.sub.2 (TXA.sub.2), which cause the formation of platelet aggregates on the damaged wall (17). These platelet aggregates are regarded as the primary hemostatic defense mechanism of the body against blood loss. However, under certain pathological conditions, due to loss or perturbation of the vascular endothelial cells which line the lumen of the vessel wall, platelet aggregates may be formed which play a major role in various clinical disorders including cardiovascular diseases, graft rejection, vascular prostheses, artificial cardiac valves and cancer (18). Following attachment to the vessel wall, platelets may release potent mitogens (platelet-derived growth factor--PDGF) contained within their granules which may stimulate the medial smooth muscle cells (SMC) to proliferate and form the primary lesions of atherosclerosis (18). Medial SMC proliferation is also the cause of the late closure of vascular grafts (e.g. coronary entry bypasses).
There are several specific receptors on platelet membranes which are involved in the formation of platelet aggregates. These receptors are activated by different agonists which are present at sites of thrombus formation. These agonists include ADP released from injured erythrocytes, collagen platelet activating factor (PAF--acether) epinephrine and thrombin. Most agonists activate platelets by binding to their specific receptor, a process which leads to hydrolysis of platelet membrane phosphatidylinositol diphosphate by phospholipase C and causes calcium mobilization from the dense tubular system (17).
Platelet activation causes the release of intrinsic substances (ADP, TXA.sub.2, serotonin) which amplify the aggregation process and influence the cells of the vessel wall. Platelet activation also initiates the activation of the clotting system which leads to the formation and polymerization of fibrin.
Thus, the observations of the involvement of the platelets in many pathological conditions led to the realization of the therapeutic importance of drugs which inhibit platelet aggregation. The efficacy of anti-aggregating agents such as aspirin or a combination of aspirin and dipyridamole has been demonstrated in patients with myocardial infarction, prosthetic heart valves and vascular grafts and in patients with organ transplants. Other drugs such as sulfinpyrazone and ticlopidine were also shown to have a beneficial effect in certain thromboembolic disorders (17).
However, though many drugs have inhibitory effects on platelet function, none of them inhibit all the mechanism's which are involved in various forms of thrombosis (19).
Aspirin, which is the most popular platelet inhibitor drug, blocks the arachidonate pathway in platelets and can be expected to be effective against thromboembolic events in which TXA.sub.2 generation plays a major role, but not if other metabolic pathways predominate, e.g. if thrombin or collagen are the major platelet activator (17). Furthermore dipyridamole, which elevates platelet cAMP levels, has not been shown to have beneficial effects on myocardial infarction when administered alone (17).
Combinations of oral anticoagulants and aspirin have only recently been tested for their antithrombotic efficacy.
Leech saliva obtained by phagostimulation of leeches with ariginine/saline has been shown to inhibit platelet aggregation induced by different agonists (12). Several substances from leech saliva may exert their inhibitory effect. Hirudin, discussed above, inhibits platelet aggregation induced by thrombin, but not by other agonists. Leech collagenase, inhibits collagen-induced aggregation. Leech apyrase inhibits ADP-induced aggregation. Fractionation of lyophilized saliva on Bio-Gel P-2 showed that high molecular weight components inhibited thrombin -, collagen -, and ADP- induced aggregation (12). These inhibitions are apparently due to the presence in this high molecular weight fraction of hirudin, collagenase and apyrase, respectively.
It has now been found that the low molecular weight fractions, obtained by fractionation of lyophilized saliva of the medicinal leech also comprise components which inhibit platelet aggregation, which components are the subject of the present invention.